CN1071791C - New DNA sequences - Google Patents

Abstract

The present invention relates to a DNA molecule comprising an intronic sequence and encoding a human protein called bile salt-stimulated lipase (BSSL) or carboxylester lipase (CEL) depending on the site of action. This DNA molecule can be used to produce recombinant human BSSL/CEL, especially from genetically mutated non-human mammals. The recombinant human BSSL/CEL can be used as an infant milk formula component to replace human milk-fed infants, or it can be used to produce drugs against malabsorption, cystic fibrosis and chronic pancreatitis.

Description

DNA molecule encoding BSSL/CEL

The present invention relates to a DNA molecule comprising an intronic sequence and encoding a human protein called bile salt-stimulated lipase (BSSL) or carboxylase lipase (CEL) depending on the site of action. The use of this DNA molecule facilitates the production of recombinant human BSSL/CEL, especially by using transgenic non-human mammals. The recombinant human BSSL/CEL can be used as a component of infant food that can replace human milk to feed infants, or it can be used to produce anti-fat absorption disorders, cystic fibrosis and chronic pancreatitis. Hydrolysis of food lipids

Lipids in food are an important source of energy. Energy-rich triacylglycerols make up over 95% of these lipids. Some lipids, such as certain fatty acids and fat-soluble vitamins, are essential components of the diet. Triacylglycerols, as well as minor constituents, namely esterified fat-soluble vitamins and cholesterol, and diacylphosphatidylglycerols, require hydrolysis of ester bonds to yield less hydrophobic absorbable products prior to gastrointestinal absorption. These reactions are known as Catalyzed by a specific group of enzymes known as lipases.

In adults, the essential lipases thought to be involved are gastric lipase, pancreatic colipase-dependent lipase (hydrolyzes tri- and diacylglycerols), pancreatic phospholipase A2 (hydrolyses diacylphosphatidylglycerols), and carboxylate lipids Enzyme (CEL) (hydrolyzes cholesteryl and fat-soluble vitamin esters). In breastfed neonates, bile salt-stimulated lipase (BSSL) plays an essential role in the hydrolysis of several of the above lipids. The products of lipid digestion form mixed micelles with bile salts to initiate absorption. bile salt stimulated esterase

The mammary glands of breastfed humans synthesize and secrete with milk bile salt-stimulated lipase (BSSL) (Blöckberg et al., 1987), which, after specific activation by primary bile salts, acts on the endogenous affect intestinal fat digestion. This enzyme (Blckberg & Hernell, 1981), which accounts for about 1% of the total milk protein content, does not decrease when it passes through the stomach with milk. Chymotrypsin is inactivated into bile salts to protect the enzyme. But when the milk is pasteurized, it can be inactivated by heating to 62.5°C for 30 minutes (Bjrksten et al., 1980).

In vitro model experiments have shown that the end products of triacylglycerol digestion differ in the presence of BSSL (Bernböck et al., 1990; Hernell & Blöckberg, 1982). This may be due to the lower intraluminal bile salt concentration that favors product absorption during the neonatal period. Carboxylate esterase

Carboxylate ester lipase (CEL) in human pancreatic juice (Lombardo et al, 1978) appears to function identically, or at least closely, to BSSL (Blöckberg et al, 1981). They have a common antigenic determinant, have the same N-terminal amino acid sequence (Abouakilet al., 1988), and are inhibited by serine esterase inhibitors, such as physostigmine and diisopropyl fluorophosphate. The cDNA structures of milk lipase and pancreatic lipase have been characterized in recent studies by several laboratories (Baba et al., 1991; Hui et al., 1991; Nilsson et al., 1990; Reue et al., 1991 ) and concluded that the milk enzyme is the product of the same gene as pancreatin (relevant to the CEL group in this application, EC 3.1.1.1). The cDNA sequence and deduced amino acid sequence of the CEL gene are described in WO 91/15234 (Oklahoma Medical Research Foundation) and WO 91/18923 (Aktiebolaget Astra).

Therefore, CEL is considered to be the same as BSSL, and the polypeptide encoded by the CEL gene is referred to as BSSL/CEL in the specification of the present invention. lipid malabsorption

A common cause of lipid malabsorption, and hence malnutrition, is reduced levels of intraluminal pancreatic colipase-dependent lipase and/or bile salts. Classic examples of lipase deficiency are patients with cystic fibrosis, 80% of patients with cystic fibrosis intercourse is a common genetic disorder leading to a lifelong defect, and chronic pancreatitis often results from chronic alcoholism.

Patients with pancreatic lipase deficiency are currently treated with oral very high doses of a crude porcine pancreatic enzyme preparation. But in the stomach, low pH inactivates coenzyme-dependent pancreatic lipase. However, the use of large doses of enzymes did not completely overcome this effect. Therefore, oral large doses are not suitable for most patients, and moreover, the preparation is impure and tastes bad.

Specific tablets have been made to pass through the acidic region of the stomach and release the enzyme only in the relatively alkaline environment of the jejunum. However, many patients with pancreatic abnormalities have an abnormally acidic jejunum, so in this case it is unlikely that the tablet will release the enzyme.

Furthermore, since the products currently on the market are not of human origin, there is a risk of an immune response that could adversely affect the patient or lead to a diminished therapeutic effect. Another disadvantage of the existing formulations is that there is no indication of lipolytic activity other than colipase-dependent lipase in their contents. In fact, most of these formulations contained very low levels of BSSL/CEL activity. This may be one reason why many patients with cystic fibrosis suffer from deficiencies in fat-soluble vitamins and essential fatty acids despite supplementation therapy.

Therefore, there is a great need for a product having the properties and structure of human lipase with broad substrate specificity which can be administered orally to patients suffering from deficiencies in one or more of the pancreatic lipolytic enzymes. The products obtained using the present invention fulfill this need by themselves or in combination with formulations containing other lipases. baby food

Everyone at home knows that human milk is the best way to feed infants and young children. Human milk not only provides well-balanced nutrition, but is also easily digested by babies. Thus, several biologically active components known to have physiological functions in infants are either components of human milk or are produced during its digestion, including components that fight infection and that facilitate the absorption of nutrients from human milk. Element.

Despite the enormous effort that has been expended in the preparation of baby food, it has not been possible to produce a product which shares the advantages of human milk to any significant extent. Therefore, marital food is usually prepared on the basis of milk, which cannot be completely digested by the baby, and lacks known substances that have an impact on the baby's physiological functions. In order to obtain infant food with a nutritional value similar to that of human milk, a large number of additions including protein fragments, vitamins, minerals, etc. (formed or absorbed during the digestion of human milk by the infant) are added to the food, which at the same time causes the increase of vital organs. Such as the burden on the liver and kidneys and the danger of possible long-term damage to them. A hazard associated with the use of cow's milk-based foods is the increased risk of inducing an allergic reaction to bovine protein in infants.

In addition to milk-based baby food, human milk obtained from so-called milk banks can also be used. However, the feeding of newborns with human milk from milk banks will not increase widely in recent years due to fears of infectious agents such as HIV and CMV present in human milk. In order to destroy the infectious agents in human milk, it must be pasteurized before use. However, pasteurization reduces the nutritional value and biological effects in the milk, eg inactivation of BSSL as described above. Adding lipase to baby food

At birth, the pancreas and liver are not fully functional, especially in babies born before term. Physiologically, fat malabsorption is commonly found and is thought to be caused by low intraluminal concentrations of pancreatic coenzyme-dependent lipase and bile salts. However, due to the presence of BSSL, the aforementioned malabsorption occurs less frequently in breastfed infants than in infants fed pasteurized human milk or infant food (Bernböck et al., 1990)

In order to avoid the above-mentioned disadvantages associated with pasteurized milk and milk-based baby foods, it is therefore desirable to prepare baby foods whose composition is similar to that of human milk, ie foods containing human milk proteins.

BSSL/CEL has several unique properties that make it ideal for use in complementary baby foods:

It is designed by nature to be taken orally. Therefore, it withstands passage through the stomach and is activated by the contents of the small intestine.

• Its specific activation mechanism prevents the deleterious lipolytic action of food or tissue lipids during storage and delivery to its site of action.

• Due to its broad substrate transtropy, it has the potential to mediate, on its own, complete digestion of most dietary lipids, including fat-soluble vitamin vinegars.

· BSSL/CEL may be better than pancreatic colipase-dependent lipase in hydrolyzing ester bonds containing long-chain polyunsaturated fatty acids.

BSSL/CEL can completely digest triacylglycerols in vitro in the presence and absence of gastric lipase, or at low levels of colipase-dependent lipase, even with low levels of bile salts, as in neonates. In the presence of BSSL/CEL, the end products of triacylglycerol digestion are free fatty acids and free glycerol, rather than free fatty acids and monoacylglycerols produced by the other two lipases (Bernböck et al., 1990). This may facilitate product absorption, especially in cases where intraluminal bile salt levels are low.

Supplementing infant formula with BSSL/CEL requires the utilization of a large number of products. Although it is possible to directly purify human milk proteins from human milk, this is not a realistic and economical route to obtain large quantities of human milk proteins needed for large-scale milk substitute production, and it is difficult to prepare infant formula containing human milk proteins Before dairying, other methods had to be developed accordingly. The present invention provides the above-mentioned method for preparing BSSL/CEL in large quantities. Protein production from milk of genetically mutated animals

Isolation of genes encoding pharmaceutically active proteins allows for the relatively inexpensive production of such proteins in heterologous systems. An attractive expression system for milk proteins is the genetically mutant animal (see review by Hennighausen et al., 1990). In EP317,355 (Oklahoma-Medical Research Foundation) a dietary composition containing a bile salt-activated lipase derived from eg genetically mutated animal technology is described.

In genetically mutant animals, protein coding sequences can be introduced as cDNA or genomic sequences. Since introns are essential for the expression of the regulated gene in mutant animals (Brinster et al., 1988; Whitelaw et al., 1991), in many cases it is better to use the genomic form rather than the structural gene the DNA form. WO 90/05188 (Pharmaeutical Proteins Limited) describes the use in genetically mutant animals of protein-encoding DNA containing at least one, but not all, introns naturally present in a protein-encoding gene.

The object of the present invention is to provide a method for producing recombinant human BSSL/CEL for infant formula with high yield and realistic price, so as to avoid the disadvantages of pasteurized milk and milk substitute based on bovine protein.

The purpose of the present invention is achieved by cloning and determining the sequence of the human CEL gene. To increase the yield of BSSL/CEL, the obtained DNA molecule containing the intron sequence of the human CEL gene has been used in place of the known cDNA sequence to produce human BSSL/CEL in genetically mutated non-human mammals.

Correspondingly, the present invention relates to the DNA molecule shown in SEQ ID NO: 1 in the Sequence Catalog, or under stringent hybridization conditions, the DNA molecule hybridized with the DNA molecule shown in SEQ ID NO: 1 in the Sequence Catalog Analogs or specific parts thereof.

Methods for isolating human BSSL/CEL DNA molecules are described in the following Examples.

Stringent hybridization conditions mentioned above are understood in their classical sense, ie hybridization is accomplished following common laboratory procedures such as Sambrook et al. (1989).

Another aspect of the present invention is to provide a mammalian expression system comprising a DNA sequence encoding human BSSL/CEL, wherein the DNA sequence is inserted into a gene encoding a milk protein of a non-human mammal to form a hybrid gene which can be expressed in a gene comprising The hybrid gene is expressed in the mammary gland of an adult female mammal so that when the hybrid gene is expressed, human BSSL/CEL is produced.

In another aspect, the present invention relates to a method for producing a non-human mammal capable of expressing a gene mutation of human BSSL/CEL, comprising injecting the above-mentioned mammalian expression system into a fertilized egg or embryonic cell of a mammal, so as to incorporate the expression system into mammalian larvae, and the resulting injected fertilized eggs or embryos develop into adult female mammals.

In the sequence directory, a DNA molecule with a full length of 11531bp, as a SEQ ID NO: 1, has the following features: Features starting alkaline base terminal 5 ＇side area 1 1640tata box 161117 outer show 1 1641 1 1641 1727 Translation starting 1653 1653 outer Xianzi 2 4071 4221 outer Xianzi 3 4307 4429 outer Xianzi 4707 4904 outer appendal 5 6193 6323 outer show 6501 6608 outer appendal 7 6751 6868 outer Xianzi 88335 8521 Display Son 9 8719 8922 outer Xianzi 10124 10321 outer Xianzi 111650 114903 ＇side area 11491 11531

In the present invention, the term "gene" is used to describe the DNA sequence that is present in the production polypeptide chain and includes the leading region as well as the subsequent coding region (5' upstream and 3' downstream sequences) and intermediate sequences called introns , introns are located between two separate coding segments (so-called exons) or in the 5' upstream or 3' downstream regions. The 5'upstream region contains regulatory sequences that control gene expression, typically a promoter. The 3' downstream region contains sequences involved in gene transcription termination and may contain sequences responsible for the polyadenylation of transcripts and the 3' untranslated region.

The DNA molecules of the invention as explained herein may include natural as well as synthetic DNA sequences, the natural sequences being generally obtained directly from the genomic DNA of normal mammals, as described below. Synthetic sequences can be prepared according to classical methods for synthetically preparing DNA molecules. The DNA sequence can also be a mixture of genomic and synthetic sequences.

In another aspect, the present invention relates to a replicable expression vector, which can carry and mediate the expression of the DNA sequence encoding human BSSL/CEL.

In the present invention, the term "replicable" means that the vector is capable of replicating in a given host cell into which it has been introduced. In cases where secretion of human BSSL/CEL expressed by host cells containing the vector is ensured, a sequence encoding a signal peptide may be provided immediately upstream of the human BSSL/CEL DNA sequence. The signal sequence may be a native signal sequence related to the human BSSL/CEL DNA sequence or a signal sequence from other sources.

The vector can be any vector capable of performing recombinant DNA operations, and the choice of the vector often depends on the host cell into which it is introduced. Thus, the vector may be a self-replicating vector, ie the vector may exist as an extrachromosomal entity whose replication is independent of chromosomal replication, examples of such vectors being plasmids, bacteriophages, cosmids, minichromosomes or viruses. In addition, the vector may be a vector that, when introduced into a host cell, is integrated into the genome of the host cell and replicated together with the chromosome into which it has been integrated. Examples of suitable vectors are bacterial expression vectors and yeast expression vectors. The vector of the present invention can carry any of the aforementioned DNA molecules of the present invention.

The invention also relates to cells containing a replicable expression vector as defined above. In principle, the cell may be any type of cell, ie, a prokaryotic cell, a unicellular eukaryote or a cell derived from a multicellular organism, such as a mammal. Mammalian cells are particularly suitable for the purposes of the present invention, as discussed further below.

In another important aspect, the present invention relates to a method for producing recombinant human BSSL/CEL, wherein, the DNA sequence encoding human BSSL/CEL is inserted into a vector capable of replicating in a specific host cell, and the resulting recombinant vector is introduced into Under suitable conditions, in a suitable medium or in host cells grown on the surface to express human BSSL/ECL and recover human BSSL/CEL.

The medium used for growing the cells may be any conventional medium suitable for the purposes of the present invention. Suitable vectors may be any of those described above, and suitable host cells may be any of the cell types listed above. The method for constructing the vector and efficiently introducing it into the host cell may be any method known in the field of recombinant DNA that can achieve the purpose of the present invention. Recombinant human BSSL/CEL expressed by cells can be secreted, ie exported through the cell membrane, but this depends on the type of cell and the composition of the vector.

If human BSSL/CEL are produced intracellularly by recombinant cells, i.e. not secreted by the cells, then it is possible to recover them by standard methods, including by mechanical means such as sonication or homogenization, or by enzymatic or chemical disruption of the cells , and then purified.

In order to be secreted, the DNA sequence encoding human BSSL/CEL should be preceded by a sequence encoding a signal peptide, in order to ensure that human BSSL/CEL is secreted from the cell, so that at least a significant proportion of the expressed human BSSL/CEL CEL is secreted into the culture medium and it is recycled.

A presently preferred method of producing recombinant human BSSL/CEL according to the invention is the use of a genetically mutated non-human mammal capable of secreting human BSSL/CEL into its milk. The advantage of using genetically mutated non-human mammals is that high yields of recombinant human BSSL/CEL can be obtained at a reasonable price, especially when the non-human mammal is a cow, the use of recombinant human BSSL/CEL as a milk-based No further purification is necessary for the production of recombinant human BSSL/CEL in milk as a normal component such as infant formula for nutritional supplementation in products. Furthermore, production in higher organisms such as non-human mammals generally results in correct processing of mammalian proteins, corresponding post-translational processing as described above, and proper folding. Large quantities of substantially purified human BSSL/CEL are also available.

Accordingly, in another important aspect, the present invention also relates to a mammalian expression system comprising a DNA sequence encoding human BSSL/CEL inserted into a non-human mammalian milk protein encoding gene to form a hybrid gene, containing said The hybrid gene can be expressed in the mammary gland of an adult female mammal of the hybrid gene.

The DNA sequence encoding human BSSL/CEL is preferably the human BSSL/CEL gene or its analogue of the DNA sequence shown in SEQ ID NO: 1 in the sequence catalog or genome.

It is generally believed that the mammary gland and the genes encoding milk proteins are particularly suitable for the production of heterologous proteins in genetically mutated non-human mammals as the expression tissue, as is the natural production of milk proteins at high expression levels in the mammary gland. In addition, milk is easy to collect and is available in large quantities. In the present invention, the use of a milk protein gene in the production of recombinant human BSSL/CEL also has the advantage that recombinant human BSSL/CEL can be produced under conditions similar to its natural production conditions in accordance with regulation of expression and production site (mammary gland) .

The term "hybrid gene" herein refers on the one hand to a DNA sequence comprising the DNA sequence encoding human BSSL/CEL as defined above, and on the other hand to a DNA sequence capable of mediating the expression of the milk protein gene DNA sequence of the hybrid gene product. The term "gene encoding milk protein" refers to the entire gene and its subsequences that can mediate and determine the expression of the hybrid gene into the desired tissue, namely mammary gland. Usually, the subsequence at least includes one or more promoter regions, transcription start sites, 3' and 5' non-coding regions and structural sequences. Preferably, the DNA sequence encoding human BSSL/CEL is substantially free of prokaryotic sequences, such as vector sequences, as related to its cloned DNA sequence.

Preferably in vitro, using techniques known in the art, the DNA sequence encoding human BSSL/CEL is inserted into the milk protein gene to form a hybrid gene. Alternatively, the DNA sequence encoding human BSSL/CEL can be inserted by homologous recombination in vivo.

Typically, the DNA sequence encoding human BSSL/CEL is inserted into one of the first exons of the milk protein of choice or contains the base of the first exon and preferably 5' flanking sequences thought to be of regulatory importance. Part of its effective subsequence.

The hybrid gene preferably contains a sequence encoding a signal peptide to enable proper secretion of the hybrid gene product into the mammary gland. Signal peptides are typically those normally found in said milk protein genes or are related to the DNA sequence encoding human BSSL/CEL. However, other signal sequences that drive secretion of the hybrid gene product into the mammary gland are also possible. Of course, the various components of the hybrid gene should also be fused in the manner described above in order to properly express and process the gene product. Therefore, usually the selected DNA sequence encoding the signal peptide should be precisely fused to the N-terminal portion of the DNA sequence encoding human BSSL/CEL. In the hybrid gene, the DNA sequence encoding human BSSL/CEL usually contains its own stop codon, but not its own message cleavage, and polyadenylation site. Downstream of the DNA sequence encoding human BSSL/CEL, mRNA processing sequences for milk proteins are generally retained.

Many factors are expected to affect the actual expression level of a particular hybrid gene. The ability of the promoter and other regulatory sequences mentioned above, the integration site of the expression system in the mammalian genome, the integration site of the DNA sequence encoding human BSSL/CEL in the milk protein coding gene, the components with the ability of post-transcriptional regulation and other similar factors all play an important role in the resulting expression levels. On the basis of understanding the influence of various factors on the expression level of hybrid genes, those skilled in the art will know how to design an expression system suitable for the purpose of the present invention.

Various milk proteins are secreted by the mammary glands. There are two main classes of milk proteins, caseins and whey proteins. The composition of milk from different species varies with respect to the quality and quantity of these proteins. Most non-human mammals produce 3 different caseins, alpha casein, beta-casein and kappa casein. The most common bovine whey proteins are alpha and beta whey proteins. The composition of milk from various sources is further described by Clark et al. (1987).

Milk protein genes that can be used are from the same species into which the expression system is to be inserted. Or it can be obtained from another species. It has been shown here that the regulatory elements that specifically express genes into the mammary gland are species-wide functional crossovers that likely arose from a common ancestor (Hennighausen et al., 1990).

Suitable examples of the milk protein gene or its effective subsequence used when constructing the expression system of the present invention are generally found in various mammalian whey proteins, such as whey acidic protein (WAP) gene, preferably from mouse , and the beta-lactoglobulin gene, preferably from sheep. Casein genes from various sources suitable for genetic mutation to produce human BSSL/CEL can also be found, such as bovine αS1 casein and murine β casein. The preferred gene of the present invention is the murine WAP gene, which was found to provide high levels of expression of a large number of foreign human proteins in the milk of different genetically mutant animals (Hennighausen et al., 1990).

Another sequence which is preferred in connection with the expression system of the present invention is the so-called expression stabilizing sequence capable of mediating a high level of expression. A large number of facts indicate that the above-mentioned stabilizing sequence exists near and downstream of the milk protein gene.

The DNA sequence encoding human BSSL/CEL inserted into the expression system of the present invention may be genomic or synthetic or any combination thereof. For satisfactory expression, some expression systems have been found to require the presence of introns and other regulatory elements (Hennighausen et al., 1990). In some cases, it may be preferable to introduce genomic constructs rather than cDNA elements as polypeptide-encoding components into vector constructs (Brinster et al.). When using cDNA-based vectors, the intron and exon structure may lead to higher steady-state mRNA levels.

In another aspect, the present invention relates to a hybrid gene comprising a DNA sequence encoding human BSSL/CEL inserted into a gene encoding a milk protein of a non-human mammal, and a DNA sequence inserted into a milk protein gene in the above-mentioned manner, so as to contain The hybrid gene is expressed in the mammary gland of an adult female mammal. Hybrid genes and their components have been discussed in detail above. In constructing the expression system of the present invention as disclosed above, the hybrid gene constitutes an important intermediate product.

In another aspect, the invention relates to non-human mammalian cells comprising the expression system defined above. Mammalian cells are preferably embryonic or prokaryotic. The expression system is suitably inserted into mammalian cells by the methods explained below and specified in the Examples below.

In another important aspect, the present invention relates to a method for producing a mutant gene capable of expressing human BSSL/CEL non-human mammals, comprising injecting the expression system of the present invention as defined above into fertilized eggs or embryonic cells of mammals, so as to express The expression system is incorporated into mammalian larvae and the resulting injected fertilized eggs or embryos are allowed to develop into adult female mammals.

Incorporation of the expression system into mammalian larvae can be accomplished using any suitable technique, as described in "Mahippulating the Mouse Embryo"; (A Laboratory Manual, Cold Spring Harbor Laboratory Perss, 1986). For example, an expression system of hundreds of molecules can be injected directly into a fertilized egg, such as a fertilized egg cell or its pronucleus, or an embryo in a mammal of choice, and the microinjected egg is then transferred into the oviduct of a pseudopregnant nursing mother , and allow it to develop. Often, not all injected eggs develop into adult females expressing human BSSL/CEL. Thus, from an optimistic point of view, half of all mammals will be males from the following methods of breeding females.

Once integrated into the larvae, the DNA sequence encoding human BSSL/CEL can be expressed at high levels to produce correctly processed and functional human BSSL/CEL in the stable germline of the mammal.

Also needed is a method of producing a genetically mutated non-human mammal capable of expressing human BSSL/CEL but substantially unable to express its own BSSL/CEL comprising (a) disrupting the mammalian The ability of the animal to express its own BSSL/CEL so as not to express the BSSL/CEL of the mammal substantially and insert the above-mentioned expression system of the present invention or the DNA sequence encoding human BSSL/CEL into the larvae of the mammal by the above-mentioned method In order to express human BSSL/CEL in the mammal; and/or (b) replace mammalian BSSL/CEL or part thereof with the above-mentioned expression system of the present invention or DNA sequence encoding human BSSL/CEL.

The ability to express BSSL/CEL in mammals can be conveniently disrupted by introducing mutations into the DNA sequence responsible for BSSL/CEL expression. The above-mentioned mutations include mutations that render the DNA sequence without a reading frame, or introduce stop codons, or delete one or more nucleotides of the DNA sequence.

Using the known theory of homologous recombination, the mammalian BSSL/CEL gene or part thereof is replaced with the above-mentioned expression system or the DNA sequence encoding human BSSL/CEL.

In another aspect, the invention relates to a genetically mutant non-human mammal produced by the method described above.

In the broadest aspect, the genetically mutant mammal of the present invention is not limited to any particular mammal, and the mammal is generally selected from the group consisting of mouse, rat, rabbit, sheep, pig, goat and cow. For large scale production of human BSSL/CEL, larger animals such as sheep, goats, pigs and especially cattle are generally preferred due to their high milk production. However, mice, rabbits and rats are also of interest because they are easier to work with and can produce genetically mutated animals more quickly than cattle.

Progeny of the above-mentioned genetically mutant animals capable of producing human BSSL/CEL are also within the scope of the present invention.

Another aspect of the invention also includes milk of a non-human mammal comprising recombinant human BSSL/CEL.

In another aspect of the present invention, the present invention relates to an infant formula containing recombinant human BSSL/CEL, especially the above-mentioned polypeptide of the present invention. Infant formulas can be prepared by adding recombinant human BSSL/CEL or polypeptides in purified or partially purified form to the normal components of infant formulas. However, it is preferred to prepare infant formula from the above-mentioned milk of the present invention, especially milk of bovine origin. Infant formula can be prepared by conventional methods and may contain any necessary additives such as minerals, vitamins and the like.

Embodiment Example 1: the genome structure of CEL gene, sequence analysis and chromosomal location

If not mentioned otherwise, standard molecular biology techniques were used (Maniatis et al., 1982, Ausubel et al., 1987; Sambrook et al., 1989). Isolation of genomic recombinants

Using various subcloned cDNA restriction fragments (Nilsson et al., 1990) as probes, [α- ³² P]dCTP was labeled with oligomeric labeling technology (Feinberg et al., 1983), and the two species were screened by plaque hybridization. Two different human genomic phage libraries, λDASH (Clonentech Laboratories Inc., Palo Alto, Ca, USA) and λEMBL-3SP6/T7 (Stratagene, La Jolla, CA, USA). Mapping, subcloning and sequencing of genomic clones

Positive clones were digested with various restriction enzymes, electrophoresed on a 1% agarose gel, and vacuum transferred (Pharmacia LKB BTG, Uppsala, Sweden) to a nylon membrane. The membranes were hybridized with various cDNA probes. Restriction fragments that hybridized to the probe were separated by isotachophoresis (fverstedt et al., 1984). Insert smaller fragments <800bp directly into M13mp18, M13mp19, M13BM20 or M13BM21 vectors and sequence them using E. coli TG1 as host cells, and use E. coli DH5α as host bacteria to subclone larger fragments into pTZ18R or pTZ19R grave bodies and further digest them. (Plasmids pS309, pS310, and pS451 used in Example 2 below were produced accordingly). In hybridization, some of the isolated fragments are also used as probes. The full nucleotide sequence was determined with Klenow enzyme by the dideoxy chain-end method (Sanger et al., 1977) and or specific oligonucleotide M13 universal sequencing primer. Sequence data were obtained from autoradiography using the software MS-Edseq described by Sjöberg et al. (1989). The sequence was analyzed with a program derived from the UWGCG software package (Devereux et al., 1984). primer extension

Total RNA was isolated from human pancreas, mammalian mammary gland and adipose tissue using the guanidinium isothiocyanate-CsCl method (Chirgwin et al, 1979). Primer extension was performed as described by Ausubel et al. (1987) using total RNA and an antisense 26-mer oligonucleotide (5'-AGGTGAGGCCCAACACAACCAGTTGC-3'), nt 33-58. Hybridization of primers to 20 μg of total RNA was done overnight at 30°C in 30 μl of 0.9M NaCl, 0.15M Hepes pH7.5 and 0.3M EDTA. After the extension reaction with reverse transcriptase, the extension products were analyzed by 6% denaturing polyacrylamide gel electrophoresis. somatic cell hybridization

DNA from 16 human-rodent somatic hybrid cell lines obtained from the NIGMS Human Genetic Mutant Cell Repository (Coriell Institute for Medical Research Camden, NJ) was used for chromosomal determination of the CEL gene. The human-mouse somatic cell hybrid GM09925 obtained via GM09940 was obtained from human fetal male fibroblasts (IMR-91) and thymidine kinase-deficient mouse cell line B-82 (Taggart et al., 1985; Mohandas et al. al., 1986) fusion. GM10324 was crossed with GM02860 with the HPRT and APRT-deficient mouse cell line A9 (Callen et al., 1986), while the hybrid GM10611 was obtained from the reverse transcription vector SP-1 infected human lymphoblastoid cell line GM07890 with Chinese hamster ovary The cell line UV-135 (Warburton et al., 1990) was obtained by fusion of minicells. The hybrid GM10095 was obtained by fusing female lymphocytes with a balanced 46,x,t(x;9)(q13;34) karyotype with the Chinese hamster cell line CHW1102 (Mohandas et al., 1979). The human chromosomal inclusions of the hybrid cell lines as determined by cytogenetic analysis as well as by Southern blot analysis and in situ hybridization analysis are listed in Table 1. High molecular weight DNAs isolated from mouse, Chinese hamster and human parental cell lines and 16 hybrid cell lines were digested with EcoRI, fractionated in 0.8% agarose gel and transferred to nylon filters. [α- ³² P]dCTP-labeled CEL cDNA probe was prepared by oligo-labeling (Feinberg and Vogelstein, 1983) and hybridized to the filter. The filters were washed in 6xSSC/0.5% SDS and 2xSSC/0.5%SDS at 65°C for 60 minutes each. polymerase chain reaction

Exons 10 and 11 were amplified with whole human genomic DNA isolated from white blood cells, DNA from somatic cell hybrids and partial positive genomic recombinants, and total RNA from lactating human breast and human pancreas. Using 2 μg of DNA, the primers used are listed in Table 2. In 100 μl volume [10mM Tris-HCl, PH8.3, 50mM KCl, 1.5mM MgCl ₂ , each 200μM dNTP, 100μg/ml gelatin, each 100pmol primer, 1.5U Taq DNA polymerase (Perkin-Elmer Cetus, Norwalk, CT , USA)] and the annealing temperature of all primer pairs was 55°C to complete 30 cycles of PCR. RNA sequences are amplified using combined complementary DNA (cDNA) and PCR methods. At 42°C, after 30 minutes, in 50mM Tris-HCl, pH8.3, 50mM KCl, 10mM MgCl ₂ , 10μg/ml BSA, 1mM dNTP each, 500ng oligo(dt) _12-13 , 40U ribonuclease inhibitor, and 200 U reverse transcriptase (MoMuLV), (BRL, Bethesda Research Laborataries, NY, USA) in 40 μl solution, a total of 10 μg RNA was used to synthesize cDNA. The cDNA was precipitated and resuspended in 25 [mu]l _H2O ; 2 [mu]l of this was amplified as described above. The amplified fragments were analyzed on a 2% agarose gel. Some of these fragments were further subcloned and sequenced. Gene structure of human CEL gene

In each genome library, 10 ⁸ recombinants were screened and several positive clones were produced in these screens, all of them were isolated and plotted. Two clones called λBSSL1 and λBSSL5A were further analyzed. Restriction enzyme digestion Southern blot analysis with several enzymes, followed by cDNA probe hybridization, showed that the λBSSL5A clone covered the entire CEL gene, and the λBSSL1 clone covered the 5'-half and 10kb of the 5' side region (Figure 1). Together, these two clones cover approximately 25 kb of the human genome.

After subcloning and restriction enzyme digestion, fragments suitable for sequencing were obtained, and the entire sequence of the CEL gene could be determined, including the 1640 bp 5' side region and the 41 bp 3' side region. These data indicate that the human CEL gene (SEQ ID NO: 1) spans a region of 9850 bp and contains 11 exons separated by 10 introns (Figure 1). This means that exons and specifically introns are quite small. In fact, exons 1-10 range in size from 87-204 bp, respectively, and exon 11 is 841 bp long. The size of the introns ranged from 85-2.343bp, respectively. It can be noted in Table 3 that all exon/intron boundaries follow the AG/GT rule and fit well with the consensus sequence indicated by Mount et al. (1982). Comparing the coding part of the CEL gene with the cDNA (Nilsson et al., 1990), only one difference was found in the nucleotide sequence, the 2nd nt in exon 1 was ac, and in the cDNA sequence it was aT. Since this position is 10 nt upstream of the translation initiation codon ATG, this difference does not affect the amino acid sequence. The seven members of the Alu class of repetitive DNA factors reside in the sequenced region, labeled Alu1-Alu7 (5'-3') (Figure 1), one of which is in the 5' side region, and the other six are in the CEL gene. Transcription start site and 5' side region

To map the transcription start sites of the human CEL gene, primer extension assays were performed using total RNA from human pancreas, lactating mammary gland, and adipose tissue. The results showed that a major transcription initiation site was located 12 bp upstream of the initiation factor methionine, while a minor initiation site was located 8 bases away. The transcription start site was identical in pancreas and lactating mammary gland, while no signal was detected in adipose tissue (Fig. 2). The sequenced region includes 1640 nt of the 5' side DNA. On the basis of sequence similarity, a TATA box-like sequence, CATAAAT, was found upstream of the transcription initiation site (Fig. 4). There are neither CAAT box structures nor GC boxes in this region.

Computer screening of the 5' side sequences of the two strands as nucleotide sequences for binding sequences of other mammary- and pancreas-specific gene transcripts revealed several putative recognition sequences, see Figure 4. Chromosomal location of CEL gene

In the human control DNA, four EcoRI fragments of about 13kb, 10kb, 2.2kb and 2.0kb were detected by the CEL cDNA probe, and a single fragment of about 25kb and 8.6kb was detected in the mouse and hamster control DNA, respectively. In hybrid clones, the presence of the human CEL gene sequence was only associated with the presence of human chromosome 9 (Table 1). Only 1 of the 1b hybrids analyzed was positive for the human CEL gene; this hybrid contained chromosome 9 as a human chromosome only. No inconsistencies were found for this chromosomal location, while at least two were found for any other chromosomal location (Table 1). To further submap the CEL gene, we utilized a human-Chinese hamster hybrid (GM10095) that retained a der(9) translocation chromosome (9pter→9q34:Xq13→Xqter) as the only human DNA. In this hybrid, no CEL gene sequence could be detected by Southern blotting, which indicated that the CEL gene remained in the 9q34-qter region. Example 2 Construction of expression vector

To construct a recombinant human CEL for production in milk from genetically mutant animals, the following protocol was used (Figure 5).

Three pTZ-based plasmids containing different parts of the human CEL gene, pS309, pS310, and pS311, were obtained by the method described above. Plasmid pS309 contains a SphI fragment, which covers the CEL gene from the 5' untranscribed region to part of the fourth intron. Plasmid pS310 contains a SacI fragment containing the CEL gene from part of the first intron to part of the sixth intron. Third, plasmid pS311 contains a BamHI fragment containing the CEL gene variant for the major part of the fifth intron and the remainder of the intron/exon structure. In this plasmid, the repeat sequence of exon 11, which normally encodes 16 repeats, was mutated to encode a truncated variant with 9 repeats.

Another plasmid, pS283, containing part of the human CEL cDNA cloned into the HindIII and SacI sites of pUC19, was used to fuse the gene sequence. A convenient restriction enzyme site, Kpn1, is also available with pS283, which is located in the CEL5' non-translated leader sequence. Plasmid pS283 was then digested with NcoI and SacI to isolate a fragment of about 2.7 kb. Plasmid pS309 was digested with NcoI and BspE1, and an approximately 2.3 kb fragment containing the 5' portion of the CEL gene was isolated. Digestion of plasmid pS310 with BspEI and SacI isolated a fragment of about 2.7 kb containing part of the middle region of the CEL gene. The three fragments were ligated and transformed into competent E. coli strain TG2, and transformants were isolated by ampicillin selection. Plasmids were prepared from a large number of transformants and one plasmid called pS312 (Figure 6) containing the desired construct was used for further experiments.

To obtain a modification of pS311 in which the BamHI site located downstream of the stop codon was changed to a SalI site for further secondary cloning, the following method was used. pS311 was linearized by partial BamHI digestion. The linearized fragment was isolated and inserted into a synthetic DNA linker converting BamHI to a SalI site (5'-GATCGTCGAC-3') and thereby disrupting the RamHI site. The resulting plasmid was analyzed by restriction enzyme cleavage since there were two potential sites for the integration of the synthetic linker. A plasmid with the adapter inserted at the desired position downstream of exon 11 was isolated and named pS313.

To obtain an expression vector construct containing the CEL genomic sequence and encoding a truncated CEL variant, a plasmid pS314 was used for an intermediate step and for tissue-specific expression in mammary cells during targeting. Plasmid pS314 contains a genomic fragment from the mouse whey acidic protein (WAP) gene (Campbell et al, 1984) cloned as a NotI fragment. This genomic fragment has about 4.5 kb of upstream regulatory sequences (URS), complete transcribable exon/intron regions and about 3 kb of downstream sequence of the last exon. The only KpnI site is located at 24 bp of the first exon upstream of the native WAP translation start codon. Another unique restriction enzyme site is the SalI site located in exon 3. In pS314, this SalI site was destroyed by digestion, filled in with klenow and religated. Instead, a new SalI site was introduced directly downstream of the KpnI site in exon 1. This protocol was completed by KpnI digestion and introduction of the annealed synthetic oligos SYM24015'-CGTCGACGTAC-3' and SYM24025'-GTCGACGGTAC-3' at this position (Figure 8). The human CEL genome sequence was inserted between these two sites KpnI and SalI by the following method. First, pS314 was digested with KpnI and SalI, and the fragments representing the cleaved plasmid were separated by electrophoresis. In the second step, pS312 was digested with KpnI and BamHI and an approximately 4.7 kb fragment of the 5' portion of the human CEL gene was isolated. In the third step, pS313 was digested with BamHI and SalI and the 3' portion of the human CEL group was isolated. The three fragments were ligated and transformed into competent E. coli bacteria, and transformants were isolated after ampicillin selection. Plasmids were prepared from several transformants and carefully analyzed by restriction enzyme mapping and sequence analysis. A plasmid representing the desired expression vector was identified and named pS317.

To construct a genomic CEL expression vector encoding full-length CEL, pS317 was modified as follows (Fig. 5). First, pTZ18R plasmid (Pharmacia), pS451 containing about 5.2 kb BamHI fragment of human CEL gene downstream from intron 5 to exon 11 was digested with HindIII and SacI. This digestion yielded an approximately 1.7 kb fragment that included the HindIII site in intron 9 to the SacI site in exon 11. In the second step, plasmid pS313 was digested with SaCl and SalI to isolate a 71 bp fragment containing the 113' portion of exon and the resulting SalI site. In the third step, the WAP/CEL recombinant gene and the remainder of the plasmid sequence were isolated from pS317 as an approximately 20 kb SalI/HindIII fragment. The 3 fragments were ligated and transformed into bacteria. Plasmids were prepared from several transformants. Plasmids were digested with various restriction enzymes and subjected to sequence analysis. A plasmid containing the desired recombinant gene was identified. The final expression vector was named pS452 (Figure 7).

To remove prokaryotic plasmid sequences, pS452 was digested with NotI. The recombinant vector element consisting of murine WAP sequences flanking the human CEL genomic fragment was then isolated by agarose electrophoresis. The isolated fragments were further purified by electroelution before injection into mouse embryos.

Recombinant WAP/CEL genes for expression in the mammary gland of genetically mutant animals are listed in FIG. 8 . deposit

The following plasmids have been deposited with DSM (Deutsche Sammlung von Mikroorganismenund Zellkulturen) according to the Budapest Treaty: Plasmid Accession No. storage date pS309 DSM 7101 1991,6,121993,2,26 pS310 DSM 7102 pS451 DSM 7498 pS452 DSM 7499 Example 3 Breeding of Gene Mutant Animals

According to Example 2, the NotI fragment was isolated from plasmid pS452. This DNA fragment contains the murine WAP promoter linked to the genomic sequence encoding human BSSL/CEL. The isolated fragments were injected at a concentration of 3 ng/μl into the pronuclei of 350C57B1/6JXCBA/2J-T ₂ embryos obtained from donor mice perfused with 5 IU serum gonadotropin from a pregnant mare for hyperovulation. obtained from mice. C57B1/6JXCBA/2J- _f1 animals were obtained from Bomholtgard Breeding and Research Ceutre LTD, Ry, Denmark. After embryos were collected from the oviduct, they were isolated from a population of cells treated with hyaluronidase in M2 medium (Hogan et al., 1986). After washing the embryos were transferred to medium M16 (Hogan et al., 1986) and kept in an incubator with 5% CO ₂ gas. Injections were accomplished in M2 droplets under light paraffin oil using a Narishigi hydraulic micromanipulator and a Nikon inverted microscope fitted with Nomarski optics. After injection, healthy-looking embryos were implanted into pseudopregnant C57B1/6JXCBA/2J- _f1 recipients intraperitoneally administered 0.37 ml of 2.5% avertin. Mice that had integrated the transgene were identified using PCR analysis of DNA from tail biopsies obtained from animals at 3 weeks of age. Positive results were confirmed by Southern blot analysis. Example 4: Expression of BSSL/CEL in Gene Mutant Mice

Genetic mutant mice were identified by analysis of DNA prepared from dissected tail samples. Tissue samples were incubated with protease kappa and extracted with phenol/chloroform. If there is heterologous introduced DNA representing fragments of the expression vector, the isolated DNA is used in a PCR with primers to amplify the specific fragments. Animals are then analyzed using DNA hybridization assays to confirm PCR data and detect possible rearrangements, the structure of the integrated vector element, and to obtain information on the copy number of the integrated vector element.

In one set of experiments, 18 mice were analyzed by two methods showing that 1 mouse carried the heterologous DNA vector element from pS452. The results of PCR analysis and hybridization experiments were identical (Fig. 9).

The identified mice carrying the vector DNA elements (founder animals) were then paired and _F1 offspring were analyzed for gene gaps in the same way.

Inject female lactating animals intraperitoneally with 2IU oxytocin, and 10 minutes later, intraperitoneally anesthetize with 0.40ml 2.5% Avertin. Through a siliconized tube, connect a milk collection device to the teat, gently massage the mammary gland, and collect the milk into a 1.5ml Eppendorf tube. The amount of milk per mouse was varied between 0.1 and 0.5, depending on the number of days of lactation.

The presence of recombinant human BSSL/CEL was analyzed by SDS-PAGE, transferred to nitrocellulose filters, and incubated with polyclonal antibodies raised against native human BSSL/CEL. The obtained results indicate that the recombinant human BSSL/CEL is expressed in the milk of the gene mutant mice. Figure 10 illustrates the presence of recombinant human BSSL/CEL in the milk of mutant mice: the band is at about 116.5.

Generation of stable genetically mutated animal germlines.

In a similar way, other genetic mutant animals such as cattle or sheep that can express human BSSL/CEL can also be prepared.

The Escherichia coli strains TG2 ps 452, TG2 ps 451, TG2 ps 310, and TG2ps 309 involved in the present invention have been respectively registered as CCTCC No: M93026, CCTCC No: M93025, CCTCC No: M93024, and CCTCC No: M93023 on June 11, 1993. Preserved in China Center for Type Culture Collection. References: Abouakil, N., Rogalska, E., Bonicel, J. & Lombardo, D.(1988): Biochim. Biophys. Acta 961, 299-308. Ausubel, F.M., Brent, R.E., Moore, D.D., Smiyh , J.A.,Seidman, J.G. and Struhl, K.: Current Protocols in Molecular Biology.(Wiley Interscience, New York 1987) Baba, T., Downs, D, Jackson, K.W., Tang, J. and Wang,C.S. (1991): Biochemistry 30, 500-510. Beato, M.(1989): Cell 56, 335-344. Bernbāck, S., Blāckberg, L. & Hernell, O.(1990): J.Clin. Invest. 221-226. Bjōrksten, B., Burman, L.G., de Chateau, P., Freorl, Kzon, B., Gothefors, L. & Hernell, O. (1980): Br. Med. J.201, 267-272. Blāckberg, L.,  ngquist, K.A, & Hernell, O.(1987): FEBS Lett. 217, 37-41. Blāckberg, L. & Hernell, O. (1981): Eur. J. Biochem116, 221-225. Blāckberg, L. Lombardo , D., Hernell, O., Guy, O. & Olivecrona, T.(1981): FEBS Lett. 136, 284-288. Boulet, A.M., Erwin, C.R. and Rutter, W.J.(1986): Proc. Natl. Acad . Sci. U.S.A. 83, 3599-3603. Brinster, R.L., Allen, J.M., Behringer, R.R., Gelinas, R.E. & Palmiter, R.D.(1988): Proc. Natl. Acad. Sci. U.S.A. 85, 836, 840. Callen D.F.(1986): Ann. Genet. 29, 235-239. Campbell, S.M., Rosen, J.M., Hennighausen, L.G., Strech-Jurk, U. and Sippel, A.E.(1984): Nucleic Acid Res.12, 8685-8697. chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J.(1979): Biochemistry 18, 5294-5299. Clark, A.J., Simons, P., Wilmut, I. and Lahte, R.(1987): TIBTECH 5, 20-24.Devereux, J., Haeberli, P. and Smithies. (1984):Nucleic Acids Res. 12, 387-395.Feinberg, A. and vogelstein, B. (1983):Anal.Biochem.132, 6 -13.Hennighausen, L., Ruiz, L. & Wall, R. (1990): Current Opinion in Biotechnology 1,74-78.Hernell, O. & Blāckberg, L.(1982): Pediatr. Res. 16,882-885 .Hogan, B., Constantini, F. andLacy, E. (1986): Manipulating the mouse embryo. A Laboratory Manual. Cold Spring Harbor Laboratory Press. Hui, D. and Kissel, J.A. (1990): Febs Lett. 276, 131-134. Lombardo, D., Guy, O. & Figarella, C. (1978): Biochim. Biophys. Acta 527, 142-149. Maniatis, T., Fritsch, E.F. & Sambrook, J.: Molecular Cloning. A Laboratory Manual. (Cold Spring Harbor, NY, 1982) Mohandas, T., Sparkes, R.S., Sparkes, M.C., Shulkin, J.D., Toomey, K.E. and Funderburk, S.J. (1979): Am. J.Hum. Genet. 31, 586-600. Mohandas, T., Heinzmann, C., Sparkes, R.S. Wasmuth, J., Edwards, P. and Lusis, A.J.(1986): Somatic Cell. Mol. Genet. 12, 89-94. Mount, S.M. (1982): Nucleic Acids Res. 10, 459-472. Nilsson, J., Blāckberg, L., Carlsson, P., Enerbāck, S., Hernell, O. and Bjursell, G.(1990): Eur. J . Biochem. 192, 543-550. Qasba, M., and Safaya, S.K.(1984): Nature 308, 377-380. Reue, K., Zambaux, J., Wong, H., Lee, G., Leete, T.H. , Ronk, M., Shively, J.E., Sternby, B., Borgstrōm, B., Ameis, D. and Schotz, M.C.(1991): J. Lipid. Res. 32, 267-276. Sambrook, J., Fritsch, E.F. and Maniatis, T.E.: Molecular Cloning. A Laboratory Manual. (Cold Spring Harbor, NY, 1989) Sanger, F., Nicklen, S. and Coulson, A.R. (1977): Proc. Natl. Acad. Sci. U.S.A. 74, 5463- 5467. Sjōberg, S., Carlsson, P., Enerbāck, S. and Bjursell, G. (1989): Comput. Appl. Biol. Sci. 5,41-46.Taggart. R.T., Mohandas, T., Shows, T.B. and Bell, G.I.(1985): Proc. Natl. Acad. sci. U.S.A. 82, 6240-6244. Warburton, D., Gersen, S., Yu, M.T., Jackson, C., Handelin, B. and Housman, D.(1990): Genomics 6, 358-366. Whitelaw et al..(1991): Transgenic Research 1, 3-13. Yu-Lee, L., Richter-Mann, L., Couch, C., Stewart , F.,Mackinlay, G. and Rosen, J.(1986): Nucleic. Acid. Res.14,1883-1902.ōfverstedt, L.G., Hammarstrōm, K., Balgobin, N.,Hjerten, S., Petterson, U. and Chattopadhyaya, J.(1984): Biochim. Biophys. Acta 782, 120-126.

Table 1 In 16 human-rodent somatic cell hybrids, the relationship between CEL sequences and human chromosomes The percentage of cells and human chromosomes ^a Chromosome 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y CEL Hybrid GM09925 74 24 0 74 76 60 82 78 0 0 4 68 6 86 78 14 98 96 46 84 0 76 0 0 -GM09927 69 83 75 77 0 93 79 73 0 82 0 0 87 79 90 93 0 8 0 0 0 -GM09929 0 0 61 59 0 43 2 49 0 0 33 49 0 59 2 0 96 0 2 31 0 0 2 0 -GM09930A 0 34 62 4 12 0 26 4 0 0 6 22 56 82 12 0 0 86 78 22 82 76 6 8 -GM09932 0 0 0 68 86 46 0 80 0 2 28 26 0 0 0 0 96 0 2 0 92 0 0 0 -GM09933 50 0 84 16 54 76 92 54 0 6 0 50 84 0 8 8 92 70 80 32 94 88 0 32 -GM09934 0 50 0 0 83 79 4 87 0 0 77 87 0 2 89 0 90 89 0 91 89 2 0 0 -GM09935A 0 0 52 10 28 12 0 0 0 84 0 722 7 0 93 59 0 9 91 71 0 0 -GM09936 0 0 0 18 0 46 70 10 0 16 34 0 2 88 2 0 100 0 44 24 0 18 0 0 -GM09937 0 0 54 38 0 62 54 70 0 2 4 0 0 4 70 60 0 96 66 0 0 0 0 0 0 -GM09938 0 0 2 88 60 88 86 4 0 0 36 92 0 80 4 0 92 0 4 80 76 60 0 2 -GM09940 0 0 46 0 0 0 84 62 0 0 0 0 0 0 62 0 100 0 0 0 0 0 0 0 -GM10324 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 90 0 -GM10567 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 98 0 0 0 0 0 0 0 0 -GM10611 0 0 0 0 0 0 0 0 69 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 +GM10095 0 0 0 0 0 0 0 0 ^94b 0 0 0 0 0 0 0 0 0 0 0 0 0 94b 0 - Dissonance

4 5 88 7 7 100 9 9 0 2 6 10 5 10 7 2 13 8 5 9 7 6 3 2

16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 ^a Usually, human chromosome ^b containing 9pter-> q34 and Xq13->qter Table 2 Primers used for DNA amplification oligonucleotide nt position ^a Amplified sequence P1: 5'-AGACCTACGCCTACCTG-3' 8492-8508 Exon 10P2: 5'-TCCAGTAGGCGATCATG-3' 8646-8662P4: 5'-GACCGATGTcCTCTTCCTGG-3' 7220-7239 Exon 10P5 with primer: 5'-CAGCCGAGTCGCCCATGTTG-3' 9016-9035 Exon ^b P6 around exon 10: 5'-ACCAAGAAGATGGGCAGCAGC-3 ' 9089-9109 Duplication P7 in exon 11: 5'-GACTGCAGGCATCTGAGCTTC-3' 9722-9742

^a Nucleotide positions are given as base numbers from exon 1. To compare nucleotide positions with SEQ ID NO: 1, 1640 bases were added to the number of columns ^b Amplified from cDNA "Exon 10" Table 3 Exon-intron structure of the CEL gene

The length of the outer sub-inrticon number nucleotide length amino acids in the outer-inner sub-containing sequence number length

Position ^a (nt) Position number 5' splice donor 3' splice acceptor: (nt)1 1-87 87 1- 25 (25) CCC GCG AAG gtaaga....gtgtctccctcgcag CTG GGC CCC Ⅰ 23432 2431-2581 151 26- 75 (50) TGG CAA G gtggga....tcctgccacctgcag GG ACC CTG Ⅱ 853 2667-2789 123 76-116 (41) AAG CAA G gtctgc....gctcccccatctcag TC TCC CGG Ⅲ 2774 3067-3264 198 117- 227 36) GCC AAA AAG gtaaac....tggttctgcccccag GTG GCT GAG Ⅵ 1427 5111-5228 118 263-301 (39) CTG GAG T gtgagt....ggctctcccacccag AC CCC ATG Ⅶ 14668 6695-6881 187 302-364 (63) GTC ACG GA gtaagc....acttgattcccccag G GAG GAC Ⅷ 1979 7079-7282 204 365-432 (68) AAT GCC AA gtgagg....gtctctcccctccag G AGT GCC IX 120110 8484-8681 198 433-498 (66) AAA ACA GG gtaaga....cttctcactctgcag G GAC CCC Ⅹ 32811 9010-9850 841 499-745 (247)

^a Nucleotide positions are given as base numbers from exon 1. For comparison of nucleotide positions with SEQ ID NO: 1, 1640 bases were added to the column count.

Description of the drawings: Figure 1 CEL gene loci. The positions and restriction enzyme maps of two partially overlapping clones λBSSL1 and λBSSL5A are indicated. The exon-intron structure and the restriction enzyme sites used are shown below. Boxes numbered -1-11 represent exons. Asp=Asp700, B=BamHI, E=EcoRI, S=SacI, Sa=SalI, Sp=SphI and X=XbaI. The position and orientation of Alu repeat elements are indicated by thick arrows. a–h indicate different subcloned fragments. Figure 2 Primer extension analysis of RNA from human lactating mammary gland, pancreas and adipose tissue. Reverse transcription of the RNA was primed with a terminal radiolabeled 26-mer polyoligonucleotide (complementary to the 33 to 58 nt position of the CEL gene). Lane A is a molecular size marker (sequencing ladder), lane B is pancreatic RNA, lane C is adipose tissue RNA, and lane D is breast RNA. Fig. 3 Dot plot analysis of the 5' side region of human CEL and rat CEL gene. Regions of homology are labeled A-H and the sequences representing these portions are written, human at the top and rat at the bottom. Fig. 4 Analysis of the 5' side sequence of human CEL gene. The putative recognition sequence is underlined or underlined representing the emphasis of the complementary strand. Bold text indicates the position of homology to rCEL (regions A-H). TATA boxes are indicated by dotted lines. There are two consensus sequences with the glucocorticoid receptor binding site, GGTACANNNTGTTCT has 80% similarity sequence (Bato, M., 1989), the first one is at -231nt position (1A) on the complementary strand, the second one at -811nt position (1B). In addition, at nt position-861(2) there is a sequence with 87% similarity to the consensus sequence AGGTCANNNNTGACCT of the estrogen receptor binding site (Beato, M., 1989). Lubon and Henninghausen (1987) have analyzed the whey acidic protein (WAP) gene promoter and 5' side sequence, and established the nuclear protein binding site of the relevant mammalian mammary gland. One of them, among the large number of milk protein genes studied, such as the rat α-lactalbumin gene (Qasba et al., 1984), and the rat α-casein gene (Yu-Lee et al., 1986) , there is an 11bp conserved sequence, AAGAAGGAAGT. In the 5' side region of the CEL gene, there is a sequence with 82% similarity to the conserved sequence at nt position -1299(3) of the complementary chain.

In a study of β-casein gene regulation, a tissue-specific mammary gland factor (MGF) was identified and its recognition sequence (ANTTCTTGGNA) identified in nuclear extracts of pregnant or lactating mice. In the 5' side region of the human CEL gene, there are two sequences, one is at nt position -368 (4A) of the complementary chain, and the other is at nt position -1095 (4B), both of them have consensus sequences with the binding site of MGF 82% similarity. In addition to these two putative MGF-binding sites in the 5' side region, there is a sequence, AGTTCTTGGCA, which has 100% identity to the consensus sequence of the MGF-binding site on the complementary strand of intron Int 275.

In addition, there are four sequences with 65% similarity to the rat pancreas-specific enhancer element GTCACCTGTGCTTTTTCCCTG (Boulet et al., 1986), one at nt position -359 (5A), and the second at nt position -718 ( 5B), the third at nt position -1140 (5C), and the last at nt position -1277 (5D). Figure 5. Method for producing plasmid pS452. See Example 2 for details. Figure 6 Schematic structure of plasmid pS312. Figure 7 Schematic structure of plasmid pS452. FIG. 8 represents the actual structural diagram of the genome structure of human BSSL/CEL naturally introduced in exon 1 of WAP gene as described in Example 2. FIG. Figure 9A. Schematic illustrating the positioning of PCR primers used to identify genetically mutant animals. The 5'primer is located in the WAP sequence starting at the upstream-148bp position of the fusion between WAP and BSSL/CEL. The 3'primer is located in the 1st BSSL/CEL intron 398bp downstream of the fusion point. B． The PCR primer sequences used. C． Agarose gels indicate a typical analysis of potential founder animal PCR assays. M: molecular weight marker. Lane 1: Control PCR product produced from plasmid pS452. Lanes 2-13: PCR reactions performed with DNA preparations from potential founder animals. Figure 10 Western blot analysis of pS452 recombinant mouse WAP/human CEL gene in milk from genetically mutant mouse species. Proteins were separated on SDS-PAGE, transferred to Immobilon membrane (Millipor), and polyclonal rabbit antibody produced by high-purity human natural CEL was used, followed by reaction with alkaline phosphatase-labeled pig anti-rabbit IgG (Dakopatts), and observed with naked eyes. Lane 1: Low molecular weight markers, 106, 80, 49.5, 32.5, 27.5 and 18.5 KDa, respectively. Lane 2: High molecular weight markers: 205, 116.5, 80 and 49.5 KDa, respectively. Lane 3: 25 ng of purified non-recombinant CEL from human milk. Lane 4: 2 [mu]l milk samples diluted 1:10 from CEL genome mutant mice. Lanes 5 and 6: 2 μl milk samples diluted 1:10 from two different non-CEL mutant mice, which served as control samples.

Sequence Catalog (1) General information:

(i) Applicant:

(A) Name: ABASTRA

(B) Street: Kvarnbergagatan 16

(C) City: Sodertalje

(E) Country: Sweden

(F) Zip Code (ZIP): S-151 85

(G) Tel: +46-8-553 26000

(H) Fax: +46-8-553 28820

(I) Telex: 19237 astra S

(ii) Title of Invention: New DNA Sequence

(iii) Number of sequences: 1

(iv) Computer-readable form:

(A) Media type: Floppy disk

(B) Computer: IBM PC compatible

(C) Operating program: PC-DOS/MS-DOS

(D) Software: Patentln Release #1.0, Version #1.25(EPO)

(ⅵ) Prior application materials:

(A) Application number: SE 9201809-2

(B) Application date: 1992, June, 11

(ⅵ) Prior application materials:

(A) Application number: SE 9201826-6

(B) Application date: 1992, June, 12

(ⅵ) Prior application materials:

(A) Application number: SE 9202088-2

(B) Application date: July, 3, 1992

(ⅵ) Prior application materials:

(A) Application number: SE 9300902-5

(B) Application date: 1992,3,19(2) Information of SEQ: Q ID NO:1:

(i) Sequence features:

(A) Length: 11531.. base pairs

(B) Type: nucleic acid

(C) chain type: double chain

(D) Topology: linear

(ii) Molecular type: DNA (genome)

(ⅵ) Source:

(A) Biology: Human

(F) Tissue Type: Breast

(ⅸ) Features:

(A) Name/key: CDS

(B) Location: Connection (1653..1727,4071..4221,4307..4429,

    4707..4904,6193..6323,6501..6608,

6751..6868,8335..8521,8719..8922,

     10124..10321,10650..11394)

(ⅸ) Features:

(A) Name/key: matte-peptide

(B) Location: Connection (1722..1727,4071..4221,4307..4429,

    4707..4904,6193..6323,6501..6608,

6751..6868,8335..8521,8719..8922,

    10124..10321,10650..11391)

(D) Other information: /EC_number=3.1.1.1

/product = "Bile salt-stimulated lipase"

(ⅸ) Features:

(A) Name/key: 5'UTR

(B) Position: 1...1..640

(ⅸ) Features:

(A) Name/Key: TATA-Signal

(B) Position: 1611...1617

(ⅸ) Features:

(A) Name/Key: Exon

(B) Position: 1641...1727

(ⅸ) Features:

(A) Name/Key: Exon

(B) Position: 4071...4221

(ⅸ) Features:

(A) Name/Key: Exon

(B) Position: 4307...4429

(ⅸ) Features:

(A) Name/Key: Exon

(B) Position: 4707...4904

(ⅸ) Features:

(A) Name/Key: Exon

(B) Position: 6193...6323

(ⅸ) Features:

(A) Name/Key: Exon

(B) Position: 6501...6608

(ⅸ) Features:

(A) Name/Key: Exon

(B) Position: 675.1...6868

(ⅸ) Features:

(A) Name/Key: Exon

(B) Location: 8335...8521

(ⅸ) Features:

(A) Name/Key: Exon

(B) Position: 8719...8922

(ⅸ) Features:

(A) Name/Key: Exon

(B) Position: 10124...10321

(ⅸ) Features:

(A) Name/Key: Exon

(B) Position: 10650...11490

(ⅸ) Features:

(A) Name/Key: 3'UTRR

(B) Position: 11491...11531

(Ⅹⅰ)序列描述：SEQ ID NO:1GGATCCCTCG AACCCAGGAG TTCAAGACTG CAGTGAGCTA TGATTGTGCC ACTGCACTCT                  60AGCCTGGGTG ACAGAGACCC TGTCTCAAAA AAACAAACAA ACAAAAAACC TCTGTGGACT                 120CCGGGTGATA ATGACATGTC AATGTGGATT CATCAGGTGT TAACAGCTGT ACCCCCTGGT                 180GGGGGATGTT GATAACGGGG GAGACTGGAG TGGGGCGAGG ACATACGGGA AATCTCTGTA       240ATCTTCCTCT AATTTTGCTG TGAACCTAAA GCTGCTCTAA AAATGTACAT AGATATAAAC       300TGGGGCCTTC CTTTCCCTCT GCCCTGCCCC AGCCCTCCCC CACCTCCTTC CTCTCCCTGC       360TGCCTCCCCT CTGCCCTCCC CTTTCCTCCT TAGCCACTGT AAATGACACT GCAGCAAAGG       420TCTGAGGCAA ATGCCTTTGC CCTGGGGCGC CCCAGCCACC TGCAGGCCCC TTATTTCCTG       480TGGCCGAGCT CCTCCTCCCA CCCTCCAGTC CTTTCCCCAG CCTCCCTCGC CCACTAGGCC       540TCCTGAATTG CTGGCACCGG CTGTGGTCGA CAGACAGAGG GACAGACGTG GCTCTGCAGG       600TCCACTCGGT CCCTGGCACC GGCCGCAGGG GTGGCAGAAC GGGAGTGTGG TTGGTGTGGG       660AAGCACAGGC CCCAGTGTCT CCTGGGGGAC TGTTGGGTGG GAAGGCTCTG GCTGCCCTCA       720CCCTGTTCCC ATCACTGCAG AGGGCTGTGC GGTGGCTGGA GCTGCCACTG AGTGTCTCGG       780TGAGGGTGAC CTCACACTGG CTGAGCTTAA AGGCCCCATC TGAAGACTTT GTTCGTCGTG       840TTCTTTCACT TCTCAGAGCC TTTCCTGGCT CCAGGATTAA TACCTGTTCA CAGAAAATAC 900GAGTCGCCTC CTCCTCCACA ACCTCACACG ACCTTCTCCC TTCCCTCCCG CTGGCCTCTT       960TCCCTCCCCT TCTGTCACTC TGCCTGGGCA TGCCCCAGGG CCTCGGCTGG GCCCTTTGTT      1020TCCACAGGGA AACCTACATG GTTGGGCTAG ATGCCTCCGC ACCCCCCCAC CCACACCCCC      1080TGAGCCTCTA GTCCTCCCTC CCAGGACACA TCAGGCTGGA TGGTGACACT TCCACACCCT      1140TGAGTGGGAC TGCCTTGTGC TGCTCTGGGA TTCGCACCCA GCTTGGACTA CCCGCTCCAC      1200GGGCCCCAGG AAAAGCTCGT ACAGATAAGG TCAGCCACAT GAGTGGAGGG CCTGCAGCAT      1260GCTGCCCTTT CTGTCCCAGA AGTCACGTGC TCGGTCCCCT CTGAAGCCCC TTTGGGGACC      1320TAGGGGACAA GCAGGGCATG GAGACATGGA GACAAAGTAT GCCCTTTTCT CTGACAGTGA      1380CACCAAGCCC TGTGAACAAA CCAGAAGGCA GGGCACTGTG CACCCTGCCC GGCCCCACCA      1440TCCCCCTTAC CACCCGCCAC CTTGCCACCT GCCTCTGCTC CCAGGTAAGT GGTAACCTGC      1500ACAGGTGCAC TGTGGGTTTG GGGAAAACTG GATCTCCCTG CACCTGAGGG GGTAGAGGGG      1560AGGGAGTGCC TGAGAGCTCA TGAACAAGCA TGTGACCTTG GATCCAGCTC CATAAATACC      1620CGAGGCCCAG GGGGAGGGCC ACCCAGAGGC TG ATG CTC ACC ATG GGG CGC CTG        1673

                                                                                    , 

-23 -20CAA CTG GTG TTG GGC CTC CTC ACC TGC TGC TGG GCA GCG GCG AGT GCC 1721GLN Leu Val Leu GLY

-15 -10-5GCG AAG GTAGCCCCCCCCCCAGGGGGGGGGGTCTGC TGCTCGCG CTCAATCAGA 1777ALA LYS1TCTGGAACT TCGGGGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCGGGGGGGGGGGGGGGGGGGGGGG

CACTCACGCT CATTTCTATG GGGACAGGTG CCAGGTAGAA CACAGGATGC   CCAATTCCAT    1897TTGAATTTCA GATAAACTGC CAAGAACTGC TGTGTAAGTA TGTCCCATGC   AATATTTGAA    1957ACAAATTTCT ATGGGCCGGG CGCAGTGGCT CACACCTGCA ATCCCACCAG   TTTGGGAGGC    2017CGAGGTGGGT   GGATCACTTG   AGGTCAGGAG   TTGGAGACCA   GCCTGGCCAA   CATC GTGAAA            2077CCCCGTCTCT   ACTAAAAATA   CAAATATTAA   TCGGGCGTGG   TGGTGGGTGC   CTGTAATCCC             2137AGCTACTCGG   GAGGCTGAGG   CAGGAGAACC   GCTTGAAGCT   GGGAGGTGGA   GATTGCGGTG             2197AGCTGAGATC   ACGCTACTGC   ACTCCAGCCT   GGGTGACAGG   GCGAGACTCT   GTCTCAAAAA             2257ATAGAAAAAG   AAAAAAATGA   AACATACTAA   AAAACAATTC   ACTGTTTACC   TGAAATTCAA             2317ATGTAACTGG GCCTCTTGAA   TTTACATTTG   CTAATCCTGG   TGATTCCACC   TACCAACCTC             2377TCTGTTGTTC   CCATTTTACA   GAAGGGGAAA   CGGGCCCAGG   GGCAGGGAGT   GTGGAGAGCA             2437GGCAGACGGG   TGGAGAGAAG   CAGGCAGGCA   GTTTGCCCAG   CATGGCACAG   CTGCTGCCTC             2497CTATTCCTGT   GCAGGAAGCT   GAAAGCCGGG   CTACTCCACA   CCCGGGTCCG   GGTCCCTCCA             2557GAAAGAGAGC   CGGCAGGCAG   GAGCTCTCTC   GAGGCATCCA   TAAATTCTAC   CCTCTCTGCC             2617TGTGAAGGAG   AAGCCACAGA   AACCCCAAGC   CCCACAGGAA   GCCGGTGTCG   GTGCCCGGCC             2677CAGTCCCTGC   CCCCAGCAGG   AGTCACACAG   GGGACCCCAG   ATCCCAACCA   CGCTGTTCTG             2737CTGCCTGCGG   TGTCTCAGGC   CCTGGGGACT   CCTGTCTCCA   CCTCTGCTGC   CTGCTCTCCA             2797

CACTCCCTGG CCCTGGGACC GGGAGGTTTG GGCAGTGGTC TTGGGCTCCT GACTCAAAGG 2857

AGAGGTCACC   TTCTTCTTGG   GCGAGCTCTT   CTTGGGGTGC   TGAGAGGCCT   TCGGCAGGTC             2917ATCACGACCC   CTCCCCATTT   CCCCACCCTG   AGGCCCTCTG   GCCAGTCTCA   ATTGCACAGG             2977GATCACGCCA   CTGGCACAAG   GAGACACAGA   TGCCTCGCAG   GGGATGCCCA   CGATGCCTGC             3037ATGTGTTGCT   TCTGGTTCCT   TTCCTCCAGT   TCCAACCGCC   GCACTCTCCC   ACACCAGTGT             3097GACAGGGGGC   CCATCACCCT   AGACTTCAGA   GGGCTGCTGG   GACCCTGGCT   GGGCCTGGGG             3157GTGTAGGGCC   ACCCTGCCCT   TCCCCACCTG   GAACCTGGCA   CAGGTGACAG   CCAGCAAGCA             3217ATGACCTGGT   CCCACCATGC   ACCACGGGAA   GAGGGAGCTG   CTGCCCAAGA   TGGACAGGAG             3277GTGGCACTGG   GGCAGACAGC   TGCTTCTCAA   CAGGGTGACT   TCAAGCCCAA   AAGCTGCCCA             3337GCCTCAGTTC   CGTCAGGGAC AGAGGGTCGGA  TGAGCACCAA   CCTCCAGGCC   CCTCGTGGGG             3397GTGGACAGCT   TGGTGCACAG   AGGCCATTTT   CATGGCACAG   GGAAGCGTGG   CGGGGGTGGG             3457AGGTGTCGTC   CCTAGGGGGT   TCTTTACCAG   CAGGGGGCTC   AGGAACTGTG   GGGACTTGGG             3517CATGGGGCCA   TCGACTTTGT   GCCCAGCCAG   CTAGGCCCTG   TGCAGGGAGA   TGGGAGGAGG             3577GAAAAGCAGG   CCCCACCCCT   CAGAAAGGAG   GAAGGTTGGT   GTGAAACATC   CCGGGTACAC             3637TGAGCATTGG   GTACACTCCT   CCCGGGAGCT   GGACAGGCCT   CCCATGTGAT   GGCAAACAGG             3697CCGACAGGAG   ACACGGCTGT   TGCTCGTCTT   CCACATGGGG   AAACTGAGGA   TCGGAGTCAA             3757AGCTGGGCGG   CCATAGCCAG   AACCCAAACC   TCCATCCCAC   CTCTTGGCCG   GCTTCCCTAG             3817TGGGAACACT   GGTTGAACCA   GTTTCCTCTA   AGATTCTGGG AGCAGGACAC   CCCCAGGGAT             3877AAGGAGAGGA   ACAGGAATCC   TAAAGCCCTG   AGCATTGCAG   GGCAGGGGGT   GCTGCCTGGG             3937TCTCCTGTGC   AGAGCTGTCC   TGCTTTGAAG   CTGTCTTTGC   CTCTGGCCAC   GCGGAGTCGG             3997CTTGCCTTGC   CCCCTCCGGA   TTCAGGCCGA   TGCGGCTTGA   GCCCCCCTGA   CCCTGCCCGT             4057GTCTCCCTCG CAG CTG GGC GCC GTG TAC ACA GAA GGT GGG TTC GTG GAA          4106

Leu Gly Ala Val Tyr Thr Glu Gly Gly Phe Val Glu

5                  10GGC GTC AAT AAG AAG CTC GGC CTC CTG GGT GAC TCT GTG GAC ATC TTC         4154Gly Val Ash Lys Lys Leu Gly Leu Leu Gly Asp Ser Val Asp Ile Phe15                  20                  25                  30AAG GGC ATC CCC TTC GCA GCT CCC ACC AAG GCC CTG GAA AAT CCT CAG 4202Lys Gly Ile Pro Phe Ala Ala Pro Thr Lys Ala Leu Glu Asn Pro Gln

35 40 45CCA CAT CCT GGC TGG CAA G GTGGGAGTGG GTGGTGCCGG ACTGGCCCTG n 4251 Tr p l ProlyHis G Pro G

50CGGCGGGGCG GGTGAGGGCG GCTGCCTTCC TCATGCCAAC TCCTGCCACC TGCAG GG 4308

Glyacc CTG AAG GC AAG AAC TTC AAG AAG AAG AGA TGC CTG CAG GCC ACC ATC 4358thr Leu Lys Ala Lysn PHE LYS Leu Gln Ala Thr Ile

55                  60                  65ACC CAG GAC AGC ACC TAC GGG GAT GAA GAC TGC CTG TAC CTC AAC   ATT       4404Thr Gln Asp Ser Thr Tyr Gly Asp Glu Asp Cys Leu Tyr Leu Asn Ile70                  75                  80                  85TGG GTG CCC CAG GGC AGG AAG CAA G GTCTGCCTCC CCTCTACTCC                 4449Trp Val Pro Gln Gly Arg Lys Gln

             90CCAAGGGACC CTCCCATGCA GCCACTGCCC CGGGTCTACT CCTGGCTTGA GTCTGGGGGC       4509TGCAAAGCTG AACTTCCATG AAATCCCACA GAGGCGGGGA GGGGAGCGCC CACTGCCGTT       4569GCCCAGCCTG GGGCAGGGCA GCGCCTTGGA GCACCTCCCT GTCTTGGCCC CAGGCACCTG       4629CTGCACAGGG ACAGGGGACC GGCTGGAGAC AGGGCCAGGC GGGGCGTCTG GGGTCACCAG       4689CCGCTCCCCC ATCTCAG  TC TCC CGG GAC CTG GCC GTT ATG ATC TGG ATC          4738

Val Ser Arg Asp Leu Pro Val Met Ile Trp Ile

95                  100TAT GGA GGC GCC TTC CTC ATG GGG TCC GGC CAT GGG GCC AAC TTC CTC         4786Tyr Gly Gly Ala Phe Leu Met Gly Ser Gly His Gly Ala Asn Phe Leu105                  110                 115                 120AAC AAC TAC CTG TAT GAC GGC GAG GAG ATC GCC ACA CGC GGA AAC GTC 4834Asn Asn Tyr Leu Tyr Asp Gly Glu Glu Ile Ala Thr Arg Gly Asn Val

125 135ATC GTG GTC ACC TTC AAC CGT GGC CCC CCC CCC CTT GGG TTC CTC 4882ile Val THR PHER ARG Val Gly Phe Leu Ser

                                                                                                                       

155GCGACCAGCA TGCTGAGCCC AGCAGGGAGA TTTTCCTCAG CACCCCTCAC CCCAAACAAC       4994CAGTGGCGGT TCACAGAAAG ACCCGGAAGC TGGAGTAGAA TCATGAGATG CAGGAGGCCC       5054TTGGTAGCTG TAGTAAAATA AAAGATGCTG CAGAGGCCGG GAGAGATGGC TCACGCCTGT       5114AATCCCAGCA CTTTAGGAGG CCCACACAGG TGGGTCACTT GAGCGCAGAA GTTCAAGACC       5174AGCCTGAAAA TCACTGGGAG ACCCCCATCT CTACACAAAA ATTAAAAATT AGCTGGGGAC             5234TGGGCGCGGC GGCTCACCTC TGTAATCCCA GCACGTTGGG AGCCCAAGGT GGGTAGATCA             5294CCTGAGGTCA GGAGTTTGAG ACCAGCCTGA CTAAAATGGA GAAACCTCTT CTCTACTAAA             5354AATACAAAAT TAGCCAGGCG TGGTGGCGCT TGCCTGTAAT CCCAGCTACT CGGGAGGCTG             5414AGGCAGGAGA ATCGCTTGAA CTCAGGAGGC GGAGGTTGCG GTGAGCCGAG ATCATGCCAC             5474TGCACTCCAG CCTGGAGAAC AAGAGTAAAA CTCTGTCTCA AAAAAAAAAA AAAlAAAAAAA            5534ATAGCCAGGC GTGGTATCTC ATGCCTCTGT CCTCAGCTAC CTGGGAGGCA GAGGTGGAAG             5594GATCGCTTGA GCCCAGGGGT TCAAAGCTGC AGTGAGCCGT GGTCGTGCCA CTGCACTCCA             5654GCCTGGGCGA CAGAGTGAGG CCCCATCTCA AAAATAAGAG GCTGTGGGAC AGACAGACAG             5714GCAGACAGGC TGAGGCTCAG AGAGAAACCA GGAGAGCAGA GCTGAGTGAG AGACAGAGAA             5774CAATACCTTG AGGCAGAGAC AGCTGTGGAC ACAGAAGTGG CAGGACACAG ACAGGAGGGA             5834CTGGGGCAGG GGCAGGAGAG GTGCATGGGC CTGACCATCC TGCCCCCGAC AAACACCACC             5894CCCTCCAGCA CCACACCAAC CCAACCTCCT GGGGACCCAC CCCATACAGC ACCGCACCCG             5954ACTCAGCCTC CTGGGACCCA CCCACTCCAG CAACCAACGT GACCTAGTCT CCTGGGACCC             6014ACccccTCCA GCACCCTACC CGACCCAGCT TCTTAGGGAC CCACCATTTG CCAACTGGGC             6074TCTGCCATGG CCCCAACTCT GTTGAGGGCA TTTCCACCCC ACCTATGCTG ATCTCCCCTC             6134CTGGAGGCCA GGCCTGGGCC ACTGGTCTCT AGCACCCCCT CCCCTGCCCT GCCCCCAG GT            6194

Gly

160AAC TAT GGC CT CT CGG GAT CAG CAC ATG GCC ATT GCT GCT TGG GTG AAG AAG 6242ASN TYR GLY Leu ARG ARG ASP GLN HIS MET ALA ILE ALA TR LY LYS ARS

165 170 175Aat ATC GCG GCC TTC GGG GGG GGG GAC CCC AAC ACG ACG CTC GGG 6290asn Ile Ala Ala PHE GLY GLY GLY ASN iLe THR Leu Phe Gly Gly Geu Ge Geu Gry GLY

180 185 190GAG TCT GGA GGT GCC AGC GTC GTC TCT CTG GTCTCGGGGGGGGGG 6343GLU Ser Ala Gly GLY Ala Ser Val Leu Gln

    195                 200AGGGCCTGCC CCACAGGTTG AGAGGAAGCT CAAACGGGAA GGGGAGGGTG GGAGGAGGAG             6403CGTGGAGCTG GGGCTGTGGT GCTGGGGTGT CCTTGTCCCA GCGTGGGGTG GGCAGAGTGG             6463GGAGCGGCCT TGGTGACGGG ATTTCTGGGT CCCGTAG ACC CTC TCC CCC TAC AAC              6518

Thr Leu Ser Pro Tyr Asn

205AAG GGC CTC ATC CGG CGA GCC ATC AGC CAG AGC GGC GTG GCC CTG AGT               6566Lys Gly Leu Ile Arg Arg Ala Ile Ser Gln Ser Gly Val Ala Leu Ser210                 215                 220                 225CCC TGG GTC ATC CAG AAA AAC CCA CTC TTC TGG GCC AAA AAG                       6608Pro Trp Val Ile Gln Lys Asn Pro Leu Phe Trp Ala Lys Lys

            230                 235GTAAACGGAG GAGGGCAGGG CTGGGCGGGG TGGGGGCTGT CCACATTTCC GTTCTTTATC             6668CTGGACCCCA TCCTTGCCTT CAAATGGTTC TGAGcCCTGA GCTCCGGCCT CACCTACCTG             6728CTGGCCTTGG TTCTGCCCCC AG GTG GCT GAG AAG GTG GGT TGC CCT GTG GGT       6780

Val Ala Glu Lys Val Gly Cys Pro Val Gly

240                 245GAT GCC GCC AGG ATG GCC CAG TGT CTG AAG GTT ACT GAT CCC CGA GCC        6828Asp Ala Ala Arg Met Ala Gln Cys Leu Lys Val Thr Asp Pro Arg Ala250                 255                 260                 265CTG ACG CTG GCC TAT AAG GTG CCG CTG GCA GGC CTG GAG  T GTGAGTAGCT 6878Leu Thr Leu Ala Tyr Lys Val Pro Leu Ala Gly Leu Glu

270                 275GCTCGGGTTG GCCCATGGGG TCTCGAGGTG GGGGTTGAGG GGGGTACTGC CAGGGAGTAC     6938TCCGGAGGAG AGAGGAAGGT GCCAGAGCTG CGGTCTTGTC CTGTCACCAA CTAGCTGGTG     6998TCTCCCCTCG AAGGCCCCAG CTGTAAGGGA GAGGGGGTGC CGTTTCTTCT TTTTTTTTGA     7058GATGGAGTCT CACTGTTGCC CAGGCTGGAG TGCAGTGTCA CGATCTCAGC TCACTGCAAC     7118CTCCACCTCC TGGGTTCAAG TGATTCTCTG ACTCAACCTC CCATGTAGCT GGGACTACAG     7178GCACATGCCA CCATGCCCAG ATAATTTTTC TGTGTGTTTA GTAGGGATGG AGTTTCATCG     7238TGTTAGCTAG GATGATCTCG GTCTTGGGAC CTCATGATCT GCCCACCTCG GCCTCCCAAA     7298GTGCTGGAAT TACAGGCGTG AGCCACTGTG CCCGGCCCCT TCTTTATTCT TATCTCCCAT     7358GAGTTACAGA CTCCCCTTTG AGAAGCTGAT GAACATTTGG GGCCCCCTCC CCCACCTCAT     7418GCATTCATAT GCAGTCATTT GCATATAATT TTAGGGAGAC TCATAGACCT CAGACCAAGA     7478GCCTTTGTGC TAGATGACCG TTCATTCATT CGTTCATTCA TTCAGCAAAC ATTTACTGAA     7538CCGTAGCACT GGGGCCCAGC CTCCAGCTCC ACTATTCTGT ACCCCGGGAA GGCCTGGGGA     7598CCCATTCCAC AAACACCTCT GCATGTCAGC CTTACCAGCT TGCTACGCTA AGGCTGTCCC     7658TCACTCATTC TTCTATGGCA ACATGCCATG AAGCCAAGTC ATCTGCACGT TTACCTGACA     7718TGAGCTCAAC TGCACGGGCT GGACAAGCCC AAACAAAGCA ACCCCCACGG CCCCGCTAGA     7778AGCAAAACCT GCTGTGCTGG GCCCAGTGAC AGCCAGGCCC CGCCTGCCTC AGCAGCCACT     7838GGGTCCTCTA GGGGCCCGTC CAGGGGTCTG GAGTACAATG CAGACCTCCC ACCATTTTTG     7898GCTGATGGAC TGGAACCCAG CCCTGAGAGA GGGAGCTCCT TCTCCATCAG TTCCCTCAGT     7958GGCTTCTAAG TTTCCTCCTT CCTGCTTCAG GCCCAGCAAA GAGAGAGAGG AGAGGGAGGG     8018GCTGCCGCTG AAGAGGACAG ATCTGGCCCT AGACAGTGAC TCTCAGCCTG GGGACGTGTG     8078GCAGGGCCTG GAGACATCTG TGATTGTCAC AGCTGGGGAG GGGGTGCTCC TGGCACCTCG     8138TGGGTCGAGG CCGGGGATGC TCTAAACATC CTACAGGGCA CAGGATGCCC CTGATGGTGC     8198AGAATCAACC CTGCCCCAAG TGTCCATAGA TCAGAGAAGG GAGGACATAG CCAATTCCAG     8258CCCTGAGAGG CAAGGGGCGG CTCAGGGGAA ACTGGGAGGT ACAAGAACCT GCTAACCTGC     8318TGGCTCTCCC ACCCAG AC CCC ATG CTG CAC TAT GTG GGC TTC GTC CCT 8366

Tyr Pro Met Leu His Tyr Val Gly Phe Val Pro

280                 285GTC ATT GAT GGA GAC TTC ATC CCC GCT GAC CCG ATC AAC CTG TAC GCC       8414Val Ile Asp Gly Asp Phe Ile Pro Ala Asp Pro Ile Asn Leu Tyr Ala290                 295                 300                 305AAC GCC GCC GAC ATC GAC TAT ATA GCA GGC ACC AAC AAC ATG GAC GGC 8462Asn Ala Ala Asp Ile Asp Tyr Ile Ala Gly Thr Asn Asn Met AsP Gly

310 320CAC ATC TTC GCC AGC AGC ATC ATC ATC AAC AAC AAG GGC AAG 8510HIS Ile PHE ALA Serle ALA Ile Asn Lysn Lysn Lysn Lysn Lysn Lysn Lysn Lysn Lys

325 330 335AAA GTC ACG GA GTAAGCAGGG GGCACAGGAC TCAGGGGCGA CCCGTGCGGG 8561Lys Val Thr Glu

    340AGGGCCGCCG GGAAAGCACT GGCGAGGGGG CCAGCCTGGA GGAGGAAGGC ATTGAGTGGA    8621GGACTGGGAG TGAGGAAGTT AGCACCGGTC GGGGTGAGTA TGCACACACC TTCCTGTTGG    8681CACAGGCTGA GTGTCAGTGC CTACTTGATT CCCCCAG G GAG GAC TTC TAC AAG       8734

Glu Asp Phe Tyr Lys

345CTG GTC AGT GAT GAA ACC ACC AAG GGG CTC AGA GGC GCC AAG AAG 8782leu Val GLU PHR Ile ThR LYS GLY Leu ARG GLY ALA LYS THR.

        350                 355                 360ACC TTT GAT GTC TAC ACC GAG TCC TGG GCC CAG GAC CCA TCC CAG GAG      8830Thr Phe Asp Val Tyr Thr Glu Ser Trp Ala Gln Asp Pro Ser Gln Glu

365 370 375Aat AAG AAG AAG ACT GTG GAC TTT GAG ACC GAG ACC GAT GTC CTC CTG 8878asn LYS LYS THR Val Val Val ASP Val Leu Phe Leu Phe Leu

380                 385                 390GTG CCC ACC GAG ATT GCC CTA GCC CAG CAC AGA GCC AAT GCC AA           8922Val Pro Thr Glu Ile Ala Leu Ala Gln His Arg Ala Asn Ala  Lys395                 400                 405GTGAGGATCT GGGCAGCGGG TGGCTCCTGG GGGCCTTCCT GGGGTGCTGC ACCTTCCAGC    8982CGAGGCCTCG CTGTGGGTGG CTCTCAGGTG TCTGGGTTGT CTCGGAAAGT GGTGCTTGAG    9042TCCCCACCTG TGCCTGCCTG ATCCACTTTG CTGAGGCCTG GCAAGACTTG AGGGCCTCTT    9102TTTACCTCCC AGCCTACAGG GCTTTACAAA CCCTATGATC CTCTGCCCTG CTCAGCCCTG    9162CACCCCATGG TCCTTCCCAC TGGAGAGTTC TTGAGCTACC TTCCATCCCC CATGCTGTGT    9222GCACTGAGAG AACACTGGAC AATAGTTTCT ATCCACTGAC TCTTATGGGC CTCAACTTTG    9282

CCCATAATTT CAGCCCACCA CCACATTAAA AATCTTCATG TAATAATAGC CAATTATAAT    9342AAAAAATAAG GCCAGACACA GTAGCTCATG CCTGTAATCC CAGCACATTG GGAGGTCAAG    9402GTGGGAGGAT CACTTGAGGT CAGGAGTCTG AGACTAGTCT GGCCAACATG GCAAAACCCC    9462ATCTCTACTA AAAATACAAA AATTATCCAG GCATGGTGGT GCATGCCTAT AATCCTAGCT    9522ACTCAGGAGG CTGAGGTAGC AGAATTGATT GACCCAGGGA GGTGGAGGTT GCAGTGAGCC    9582GAGATTACGC CACTGCACTC CAGCAGGGGC AACAGAGTGA GACTGTGTCT CGAATAAATA    9642AGTAAATAAA TAATAAAAAT AAAAAATAAG TTAGGAATAC GAAAAAGATA GGAAGATAAA    9702AGTATACCTA GAAGTCTAGG ATGAAAGCTT TGCAGCAACT AAGCAGTACA TTTAGCTGTG    9762AGCCTCCTTT CAGTCAAGGC AAAAAGGGAA ACAGTTGAGG GCCTATACCT TGTCCAATCT    9822AATTGAAGAA TGCACATTCA CTTGGAGAGC AAAATATTTC TTGATACTGA ATTCTAGAAG    9882GAAGGTGCCT CACAATGTTT TGTGGAGGTG AAGTATAAAT TCAGCTGAAA TTGTGGAACC     9942CATGAATCCA TGAATTTGGT TCTCAGCTTT CCCTTCCCTG GGTGTAAGAA GCCCCATCTC    10002TTCATGTGAA TTCCCCAGAC ACTTCCCTGC CCACTGCCCG GGACCTCCCT CCAAGTCCGG    10062TCTCTGGGCT GATCGGTCCC CAGTGAGCAC CCTGCCTACT TGGGTGGTCT CTCCCCTCCA    10122G G AGT GCC AAG ACC TAC GCC TAC CTG TTT TCC CAT CCC TCT CGG ATG 10169

Ser Ala Lys Thr Tyr Ala Tyr Leu Phe Ser His Pro Ser Arg Met

410                 415                 420CCC GTC TAC CCC AAA TGG GTG GGG GCC GAC CAT GCA GAT GAC ATT CAG      10217Pro Val Tyr Pro Lys Trp Val Gly Ala Asp His Ala Asp Asp Ile Gln425                 430                 435                 440TAC GTT TTC GGG AAG CCC TTC GCC ACC CCC ACG GGC TAC CGG CCC CAA 10265Tyr Val Phe Gly Lys Pro Phe Ala Thr Pro Thr Gly Tyr Arg Pro Gln

445 450 455GAC AGG ACA GTC TCT AAG GCC ATC GCC GCC TAC TGG ACC AAC TTT GCC 10313ASP ARG THR Val Sero Met Ile Ala Tyr THR Asn PHE ALA

460 465 470AAA ACA GG GTAAGACGTG GGTTGAGTGC AGGGCGGAGG GCCACAGCCG 10361Lys Thr Gly

    475AGAAGGGCCT CCCACCACGA GGCCTTGTTC CCTCATTTGC CAGTGGAGGG ACTTTGGGCA      10421AGTCACTTAA CCTCCCCCTG CATCGGAATC CATGTGTGTT TGAGGATGAG AGTTACTGGC      10481AGAGCCCCAA GCCCATGCAC GTGCACAGCC AGTGCCCAGT ATGCAGTGAG GGGCATGGTG      10541CCCAGGGCCA GCTCAGAGGG CGGGGATGGC TCAGGCGTGC AGGTGGAGAG CAGGGCTTCA      10601GCCCCCTGGG AGTCCCCAGC CCCTGCACAG CCTCTTCTCA CTCTGCAG G GAC CCC         10656

                                                   Asp ProAAC ATG GGC GAC TCG GCT GTG CCC ACA CAC TGG GAA CCC TAC ACT ACG        10704Asn Met Gly Asp Ser Ala Val Pro Thr His Trp Glu Pro Tyr Thr Thr

480 485 490GAA AGC GGC GGC TAC CTG GAG ACC AAG AAG AAG AAG GGC AGC AGC AGC TCC 10752GLU Asn Sergr Leu GLU

495                 500                 505ATG AAG CGG AGC CTG AGA ACC AAC TTC CTG CGC TAC TGG ACC CTC ACC        10800Met Lys Arg ser Leu Arg Thr Asn Phe Leu Arg Tyr Trp Thr Leu Thr510                 515                 520                 525TAT CTG GCG CTG CCC ACA GTG ACC GAC CAG GAG GCC ACC CCT GTG CCC 10848Tyr Leu Ala Leu Pro Thr Val Thr Asp Gln Glu Ala Thr Pro Val Pro

530 535 540CCC ACA GGG GAC TCC GCC GCC ACT CCC CCC CCC CCC ACG GGT GAC TCC 10896Pro ThR GLY As GLU ALA THR PRO PRLY ASPR GLY ASSP that PLR's Pro's Pro

545 550 555GAG ACC GCC CCC GTG CCG CCC ACG GGT GCC GCC CCC CCC CCC CCC GTG 10944GLU ThR Ala Val Pro ThR Gly Ala Pro Pro Pro Val

560 565 570ccg CCC ACG GGT GAC GCC GCC CCC CCC CCC GTG CCC ACG GGT GAC 10992PRO Pro Thr Gly Ala Pro Val Pro Pro THR GLY ASP

575                 580                     585TCC GGG GCC CCC CCC GTG CCG CCC ACG GGT GAC TCC GGG GCC CCC CCC        11040Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro590                 595                 600             605GTG CCG CCC ACG GGT GAC TCC GGG GCC CCC CCC GTG CCG CCC ACG GGT 11088Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly

610 615 620GAC TCC GGG GCC CCC CCC CCC GTG CCC ACG GGT GCC GCC GCC CCC CCC 11136asp Sergial Ala Pro Val Pro ThR Gly Ala Pro Pro Pro

625 635CCC GCC ACG GGT GCC GCC GCC CCC CCC CCC GTG CCC ACG 11184PRO VAL PRO THR GLY ALA GLY Pro Pro Val Pro VAL PRO VAL Pro VAL PRO VAL PRO VAL PRO Val PRO Val PRO Val Pro Pro VR

640 645 650GGT GAC GCC GCC CCC CCC CCC GTG CCC ACG GGT GAC GCC GCC 11232GLY ASP Ser GLY ALA Pro Val Pro Thr Gly Ala

655                 660                 665CCC CCC GTG ACC CCC ACG GGT GAC TCC GAG ACC GCC CCC GTG CCG CCC        11280Pro Pro Val Thr Pro Thr Gly Asp Ser Glu Thr Ala Pro Val Pro Pro670                 675                  680                685ACG GGT GAC TCC GGG GCC CCC CCT GTG CCC CCC ACG GGT GAC TCT GAG 11328Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Glu

690 695 700GCT GCC CCT GTG CCC CCC ACA GAC TCC AAG GAA GCT CAG CCT 11376ALA Ala Val Pro Pro THR ASP Ser La Gln Met Pro

705 710 715GCA GTC ATT AGG TTT TAGCGTCCCA TGAGCCTTGG TATCAAGAGG CCACAAGAGT 11431Ala Val Ile Arg Phe

720GGGACCCCAG GGGCTCCCCT CCCATCTTGA GCTCTTCCTG AATAAAGCCT CATACCCCCTG 11491TCGGTGTCTT TCTTTGCTCC CAAGGCTAAG CTGCAGGATC 11531

Claims (4)

1. DNA molecules encoding BSSL/CEL with biological functions and containing intron sequences. 2. The DNA molecule according to claim 1, which is listed as SEQ ID No: 1 in the sequence catalogue. 3. The analog of the DNA molecule according to claim 2, which contains an intron sequence, and under stringent hybridization conditions, hybridizes with the DNA sequence shown in SEQ ID No: 1 or a specific part thereof in the sequence catalog. 4. A method for producing human BSSL/CEL, comprising (a) inserting any one of the DNA molecules defined in claims 1-3 into a vector capable of replicating in a specific host cell; (b) introducing the resulting recombinant vector into the host cell; (c) producing the resulting cells in or on culture medium to express the polypeptide; and (d) recovering the polypeptide.

Images